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1.
Arch Virol ; 165(5): 1249-1252, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32232672

RESUMO

In this study, the complete genomic sequence of a novel virus was determined by next-generation sequencing of a sample from a symptomatic strawberry plant with severe yellow spots and mosaic on its leaves. Its genomic organization and sequence showed that this virus is related to members of the proposed insect-specific genus "Negevirus". The sample also contained sequences from the geranium aphid Acyrthosiphon malvae. Although the virus was detected repeatedly in the same plant during the three following years, no other positive samples were obtained from the surroundings or more-distant locations. Reverse transcription qPCR analysis revealed the presence of both genomic positive and complementary negative strands of the viral genome in the sample, with a 3- to 30-fold excess of the positive strand, indicating active viral replication. As the virus was not detected in any insect species collected at this location, the virus was provisionally named "Fragaria vesca-associated virus 1" (FVaV-1).


Assuntos
Fragaria/virologia , Genoma Viral , Vírus de Plantas/classificação , Vírus de Plantas/isolamento & purificação , Análise de Sequência de DNA , Animais , Afídeos/genética , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Doenças das Plantas/virologia , Folhas de Planta/virologia , Vírus de Plantas/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real
2.
Arch Virol ; 165(5): 1225-1229, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32146505

RESUMO

Using a high-throughput sequencing approach, we identified four genomoviruses (family Genomoviridae) associated with a sweet orange (Citrus sinensis) plant collected in Tunisia. The ssDNA genomes of these genomoviruses, which were amplified, cloned and Sanger sequenced, range in size from 2156 to 2191 nt. Three of these viruses share > 99% full-genome pairwise sequence identity and are referred to as citrus Tunisia genomovirus 1 (CTNGmV-1). The CTNGmV-1 isolates share < 62% genome-wide pairwise nucleotide sequence identity with other genomoviruses and belong to the genus Gemykolovirus. The genome of the fourth virus, which was called CTNGmV-2, shares < 68% nucleotide sequence identity with other genomoviruses and belongs to the genus Gemycircularvirus. Based on the species demarcation criteria for members of the family Genomoviridae, CTNGmV-1 and -2 would each represent a new species. Although found associated with Citrus sp. plants, it is likely that these viruses infect fungi or other organisms associated with the plants.


Assuntos
Citrus/virologia , Vírus de DNA/classificação , Vírus de DNA/isolamento & purificação , Micovírus/classificação , Micovírus/isolamento & purificação , Análise de Sequência de DNA , Vírus de DNA/genética , Micovírus/genética , Filogenia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Tunísia
3.
Gene ; 741: 144522, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32145329

RESUMO

Virus-induced gene silencing (VIGS) is a transient based reverse genetic tool used to elucidate the function of novel gene in N. benthamiana. In current study, 14 UDP-D-glucuronate 4-epimerase (GAE) family members were identified and their gene structure, phylogeny and expression pattern were analyzed. VIGS system was optimized for the functional characterization of NbGAE6 homologous genes in N. benthamiana. Whilst the GAE family is well-known for the interconversion of UDP-D-GlcA and UDP-D-GalA during pectin synthesis. Our results revealed that the downregulation of these genes significantly reduced the amount of GalA in the homogalacturunan which is the major component of pectin found in primary cell wall. Biphenyl assay and high performance liquid chromatography analysis (HPLC) depicted that the level of 'GalA' monosaccharide reduced to 40-51% in VIGS plants as compared to the wild type plants. Moreover, qRT-PCR also confirmed the downregulation of the NbGAE6 mRNA in VIGS plants. In all, this is the first comprehensive study of the optimization of VIGS system for the provision of rapid silencing of GAE family members in N. benthamiana, eliminating the need of stable transformants.


Assuntos
Proteínas de Arabidopsis/genética , Carboidratos Epimerases/genética , Parede Celular/metabolismo , Pectinas/genética , Tabaco/genética , Arabidopsis/genética , Parede Celular/genética , Parede Celular/virologia , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Vetores Genéticos/genética , Monossacarídeos/metabolismo , Pectinas/biossíntese , Peptídeos , Vírus de Plantas/genética , RNA Mensageiro/genética , Tabaco/virologia
4.
Nucleic Acids Res ; 48(6): 3134-3155, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32083649

RESUMO

While G/U pairs are present in many RNAs, the lack of molecular studies to characterize the roles of multiple G/U pairs within a single RNA limits our understanding of their biological significance. From known RNA 3D structures, we observed that the probability a G/U will form a Watson-Crick (WC) base pair depends on sequence context. We analyzed 17 G/U pairs in the 359-nucleotide genome of Potato spindle tuber viroid (PSTVd), a circular non-coding RNA that replicates and spreads systemically in host plants. Most putative G/U base pairs were experimentally supported by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). Deep sequencing PSTVd genomes from plants inoculated with a cloned master sequence revealed naturally occurring variants, and showed that G/U pairs are maintained to the same extent as canonical WC base pairs. Comprehensive mutational analysis demonstrated that nearly all G/U pairs are critical for replication and/or systemic spread. Two selected G/U pairs were found to be required for PSTVd entry into, but not for exit from, the host vascular system. This study identifies critical roles for G/U pairs in the survival of an infectious RNA, and increases understanding of structure-based regulation of replication and trafficking of pathogen and cellular RNAs.


Assuntos
Vírus de Plantas/genética , RNA não Traduzido/genética , RNA Viral/genética , Viroides/genética , Genoma Viral/genética , Mutação , Conformação de Ácido Nucleico , Vírus de Plantas/patogenicidade , Solanum tuberosum/virologia , Viroides/patogenicidade , Viroses/genética , Viroses/virologia , Replicação Viral/genética
5.
Phytopathology ; 110(1): 68-79, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31631806

RESUMO

High-throughput sequencing technologies were used to identify plant viruses in cereal samples surveyed from 2012 to 2017. Fifteen genome sequences of a tenuivirus infecting wheat, oats, and spelt in Estonia, Norway, and Sweden were identified and characterized by their distances to other tenuivirus sequences. Like most tenuiviruses, the genome of this tenuivirus contains four genomic segments. The isolates found from different countries shared at least 92% nucleotide sequence identity at the genome level. The planthopper Javesella pellucida was identified as a vector of the virus. Laboratory transmission tests using this vector indicated that wheat, oats, barley, rye, and triticale, but none of the tested pasture grass species (Alopecurus pratensis, Dactylis glomerata, Festuca rubra, Lolium multiflorum, Phleum pratense, and Poa pratensis), are susceptible. Taking into account the vector and host range data, the tenuivirus we have found most probably represents European wheat striate mosaic virus first identified about 60 years ago. Interestingly, whereas we were not able to infect any of the tested cereal species mechanically, Nicotiana benthamiana was infected via mechanical inoculation in laboratory conditions, displaying symptoms of yellow spots and vein clearing evolving into necrosis, eventually leading to plant death. Surprisingly, one of the virus genome segments (RNA2) encoding both a putative host systemic movement enhancer protein and a putative vector transmission factor was not detected in N. benthamiana after several passages even though systemic infection was observed, raising fundamental questions about the role of this segment in the systemic spread in several hosts.


Assuntos
Genoma Viral , Vírus do Mosaico , Vírus de Plantas , Animais , Grão Comestível/virologia , Genoma Viral/genética , Hemípteros/virologia , Vírus do Mosaico/genética , Noruega , Doenças das Plantas/virologia , Vírus de Plantas/genética , Suécia
6.
Arch Virol ; 165(1): 21-31, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31624917

RESUMO

To obtain insight into the sequence diversity of strawberry latent ringspot virus (SLRSV), isolates from collections and diagnostic samples were sequenced by high-throughput sequencing. For five SLRSV isolates, the complete genome sequences were determined, and for 18 other isolates nearly complete genome sequences were determined. The sequence data were analysed in relation to sequences of SLRSV and related virus isolates available in the NCBI GenBank database. The genome sequences were annotated, and sequences of the protease-polymerase (Pro-Pol) region and coat proteins (CPs) (large and small CP together) were used for phylogenetic analysis. The amino acid sequences of the Pro-Pol region were very similar, whereas the nucleotide sequences of this region were more variable. The amino acid sequences of the CPs were less similar, which was corroborated by the results of a serological comparison performed using antisera raised against different isolates of SLRSV. Based on these results, we propose that SLRSV and related unassigned viruses be assigned to a new genus within the family Secoviridae, named "Stralarivirus". Based on the phylogenetic analysis, this genus should include at least three viruses, i.e., SLRSV-A, SLRSV-B and lychnis mottle virus. The newly generated sequence data provide a basis for designing molecular tests to screen for SLRSV.


Assuntos
Fragaria/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Secoviridae/classificação , Análise de Sequência de RNA/métodos , Proteínas do Capsídeo/genética , RNA Polimerases Dirigidas por DNA/genética , Variação Genética , Anotação de Sequência Molecular , Peptídeo Hidrolases/genética , Filogenia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , RNA Viral/genética , Secoviridae/genética , Secoviridae/isolamento & purificação
7.
Phytopathology ; 110(1): 215-227, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31483225

RESUMO

Poa semilatent virus (PSLV), Lychnis ringspot virus (LRSV), and Barley stripe mosaic virus (BSMV) are members of the genus Hordeivirus in the family Virgaviridae. However, the biological properties and molecular genetics of PSLV have not been compared with other hordeiviruses. Here, we have constructed an infectious cDNA clone of the PSLV Canadian strain and provided evidence that PSLV differs from BSMV and LRSV. First, unlike the other two hordeiviruses that replicate in chloroplasts, PSLV induces dramatic structural changes in peroxisome during its infection in barley. The αa replication protein also localizes to peroxisomes, suggesting that PSLV replication occurs in peroxisomes. Second, PSLV encodes a γb protein that shares 19 to 23% identity with those of other hordeiviruses, and its activity as a viral suppressor of RNA (VSR) silencing is distinct from those of BSMV and LRSV. Substitution of the BSMV γb protein with that of PSLV or LRSV revealed a negative correlation between VSR activity and symptom severity of the recombinant BSMV derivatives. Intriguingly, the Ser-Lys-Leu (SKL) peroxisome-targeting signals differ among γb proteins of various hordeiviruses, including some BSMV strains. The presence of the C-terminal SKL motif in the γb protein impairs its silencing suppressor activity and influences symptoms. Finally, we developed a PSLV-based virus-induced gene silencing vector that induced strong and effective silencing phenotypes of endogenous genes in barley, wheat, and millet. Our results shed new light on hordeivirus pathogenesis and evolution, and provide an alternative tool for genomics studies of model hosts and economically important monocots.


Assuntos
Hordeum , Doenças das Plantas , Vírus de Plantas , Vírus de RNA , RNA Viral , Proteínas Virais , Canadá , DNA Complementar/genética , Hordeum/virologia , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de Plantas/patogenicidade , Vírus de RNA/genética , Vírus de RNA/patogenicidade , Virulência/genética
8.
Virol J ; 16(1): 153, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31818304

RESUMO

BACKGROUND: Publicly available transcriptomic datasets have become a valuable tool for the discovery of new pathogens, particularly viruses. In this study, several coding-complete viral genomes previously not found or experimentally confirmed in alfalfa were identified in the plant datasets retrieved from the NCBI Sequence Read Archive. METHODS: Publicly available Medicago spp. transcriptomic datasets were retrieved from the NCBI SRA database. The raw reads were first mapped to the reference genomes of Medicago sativa and Medigago truncatula followed by the alignment of the unmapped reads to the NCBI viral genome database and de novo assembly using the SPAdes tool. When possible, assemblies were experimentally confirmed using 5'/3' RACE and RT-PCRs. RESULTS: Twenty three different viruses were identified in the analyzed datasets, of which several represented emerging viruses not reported in alfalfa prior to this study. Among them were two strains of cnidium vein yellowing virus, lychnis mottle virus and Cactus virus X, for which coding-complete genomic sequences were obtained by a de novo assembly. CONCLUSIONS: The results improve our knowledge of the diversity and host range of viruses infecting alfalfa, provide essential tools for their diagnostics and characterization and demonstrate the utility of transcriptomic datasets for the discovery of new pathogens.


Assuntos
Genoma Viral , Medicago sativa/virologia , Vírus de Plantas/genética , Transcriptoma , Biologia Computacional , Mineração de Dados , Bases de Dados de Ácidos Nucleicos , Medicago sativa/genética , Doenças das Plantas/virologia
9.
Genes (Basel) ; 10(11)2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31689985

RESUMO

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in Laodelphax striatellus (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable L. striatellus reference genes are still very limited. L. striatellus is a destructive rice pest and it can transmit multiple viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in L. striatellus at different developmental stages, in different tissues, in RBSDV- or RSV-infected L. striatellus or in RBSDV-infected and Lssynaptojanin 1 (LsSYNJ1)-silenced L. striatellus. The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 (LsRPL5) and LsRPL8 are the most stably expressed genes in L. striatellus at different developmental stages. Alpha-1-tubulin (Lsα-TUB) is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) L. striatellus. LsRPL8 is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) L. striatellus, while beta-tubulin (Lsß-TUB) is the most stably expressed reference gene in RBSDV-V and LsSYNJ1-silenced L. striatellus. The selected reference genes were further investigated during analyses of RBSDV P5-1 and P10 gene expression in different tissues from RBSDV-V or RBSDV-NV L. striatellus. The stably expressed reference genes identified in this study will benefit future gene function studies using L. striatellus.


Assuntos
Perfilação da Expressão Gênica/normas , Hemípteros/genética , Transcriptoma/genética , Animais , Insetos Vetores/virologia , Doenças das Plantas/virologia , Vírus de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reoviridae/genética
10.
Phys Rev Lett ; 123(13): 138101, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31697512

RESUMO

Multipartite viruses have a genome divided into different disconnected viral particles. A majority of multipartite viruses infect plants; very few target animals. To understand why, we use a simple, network-based susceptible-latent-infectious-recovered model. We show both analytically and numerically that, provided that the average degree of the contact network exceeds a critical value, even in the absence of an explicit microscopic advantage, multipartite viruses have a lower threshold to colonizing network-structured populations compared to a well-mixed population. We further corroborate this finding on two-dimensional lattice networks, which better represent the typical contact structures of plants.


Assuntos
Modelos Biológicos , Vírus de Plantas/fisiologia , Viroses/transmissão , Viroses/virologia , Genoma Viral , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírion/genética , Vírion/fisiologia
11.
PLoS One ; 14(11): e0224569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31697693

RESUMO

Rice black-streaked dwarf virus (RBSDV), a ds-RNA virus in Fijivirus genus with family Reoviridae, which is transmitted by the small brown planthopper, is responsible for incidence of maize rough dwarf disease (MRDD) and rice black-streaked dwarf disease (RBSDD). To understand the variation and evolution of S5, a unique fragment in the genome of RBSDV which encodes two partially overlapping ORFs (ORF5-1 and ORF5-2), we analyzed 127 sequences from maize and rice exhibiting symptoms of dwarfism. The nucleotide diversity of both ORF5-1 (π = 0.039) and ORF5-2 (π = 0.027) was higher than that of the overlapping region (π = 0.011) (P < 0.05). ORF5-2 was under the greatest selection pressure based on codon bias analysis, and its activation was possibly influenced by the overlapping region. The recombinant fragments of three recombinant events (14NM23, 14BM20, and 14NM17) cross the overlapping region. Based on neighbor-joining tree analysis, the overlapping region could represent the evolutionary basis of the full-length S5, which was classified into three main groups. RBSDV populations were expanding and haplotype diversity resulted mainly from the overlapping region. The genetic differentiation of combinations (T127-B35, T127-J34, A58-B35, A58-J34, and B35-J34) reached significant or extremely significant levels. Gene flow was most frequent between subpopulations A58 and B35, with the smallest |Fst| (0.02930). We investigated interactions between 13 RBSDV proteins by two-hybrid screening assays and identified interactions between P5-1/P6, P6/P9-1, and P3/P6. We also observed self-interactive effects of P3, P6, P7-1, and P10. In short, we have proven that RBSDV populations were expanding and the overlapping region plays an important role in the genetic variation and evolution of RBSDV S5. Our results enable ongoing research into the evolutionary history of RBSDV-S5 with two partly overlapping ORFs.


Assuntos
Genoma Viral/genética , Fases de Leitura Aberta/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Variação Genética/genética , Haplótipos/genética , Oryza/genética , Oryza/virologia , Filogenia , Doenças das Plantas/genética , Vírus de Plantas/patogenicidade , RNA Viral/genética , Seleção Genética
12.
Biomed Res Int ; 2019: 4630891, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781617

RESUMO

Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.


Assuntos
Bromovirus/genética , Técnicas de Transferência de Genes , Vírus de Plantas/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Células Eucarióticas/virologia , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Transfecção/métodos , Montagem de Vírus/genética
13.
Comput Biol Chem ; 83: 107127, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31542706

RESUMO

In order to maintain a consistent supply of rice globally, control of pathogens affecting crop production is a matter of due concern. Rice yellow mottle virus(RYMV) is known to cause a variety of symptoms which can result in reduced yield. Four ORFs can be identified in the genome of RYMV encoding for P1 (ORF1), Polyprotein (processed to produce VPg, protease, helicase, RdRp4) (ORF2), putative RdRp (ORF3) and capsid/coat protein (ORF4). This research was aimed at identifying genome encoded miRNAs of O. sativa that are targeted to the genome of Rice Yellow Mottle Virus (RYMV). A consensus of four miRNA target prediction algorithms (RNA22, miRanda, TargetFinder and psRNATarget) was computed, followed by calculation of free energies of miRNA-mRNA duplex formation. A phylogenetic tree was constructed to portray the evolutionary relationships between RYMV strains isolated to date. From the consensus of algorithms used, a total of seven O. sativa miRNAs were predicted and conservation of target site was finally evaluated. Predicted miRNAs can be further evaluated by experiments involving the testing of the success of in vitro gene silencing of RYMV genome; this can pave the way for development of RYMV resistant rice varieties in the future.


Assuntos
Inativação Gênica , Genoma Viral/genética , MicroRNAs/genética , Oryza/genética , Oryza/virologia , Vírus de Plantas/genética
15.
Mol Cell Probes ; 47: 101438, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31422073

RESUMO

In this study, a new SYBR Green qPCR (qRT-PCR) and a nested RT-PCR (nRT-PCR) were developed to detect Potato mop-top virus (PMTV) in potato tuber tissues. The SYBR Green qRT-PCR and nRT-PCR assays were approximately 104- and 103- fold more sensitive than the conventional RT-PCR assay. The progeny tubers derived from PMTV-infected potato tubers were tested by conventional RT-PCR, SYBR Green qRT-PCR and nRT-PCR assays. Of the 17 samples, 9 (52.9%) were positive for PMTV by conventional RT-PCR, 11 (64.7%) were positive by nRT-PCR, and 17 (100%) were positive by SYBR Green qRT-PCR. Compared to nRT-PCR, SYBR Green qRT-PCR was showed to be more sensitive. The progeny plants exhibited foliar symptoms including chlorosis and reduction in leaf size when the PMTV-positive tubers were planted in a growth chamber at 20-22 °C. These findings suggest that PMTV has been passed on to the progeny plants and tubers.


Assuntos
Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Solanum tuberosum/crescimento & desenvolvimento , Tamanho do Órgão , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/virologia , Vírus de Plantas/genética , RNA Viral/genética , Sensibilidade e Especificidade , Solanum tuberosum/virologia
16.
Arch Virol ; 164(11): 2805-2810, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31451963

RESUMO

Physalis peruviana is a perennial solanaceous plant that has recently been established as a commercial crop in Brazil. This work reports the near-complete genome sequence, particle morphology, and plant host responses to a putative new sobemovirus, named "physalis rugose mosaic virus". The virus, characterized by isometric particles of ca. 30 nm in diameter, causes foliar symptoms of mosaic, malformation and blistering, accompanied by stunting. The near-complete genome sequence comprises 4175 nucleotides and contains five open reading frames that are similar to those of other sobemoviruses. In addition to P. peruviana, the new virus systemically infected Capsicum annuum, Nicotiana tabacum and Solanum lycopersicum by mechanical inoculation. Thus, this virus may cause disease in these crops in the field.


Assuntos
Genoma Viral/genética , Vírus do Mosaico/classificação , Vírus do Mosaico/crescimento & desenvolvimento , Physalis/virologia , Doenças das Plantas/virologia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Brasil , Capsicum/virologia , Lycopersicon esculentum/virologia , Vírus do Mosaico/genética , Vírus de Plantas/crescimento & desenvolvimento , RNA Viral/genética , Tabaco/virologia
17.
Int J Mol Sci ; 20(16)2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31443292

RESUMO

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and ß-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.


Assuntos
Agrobacterium/genética , Inativação Gênica/fisiologia , Manihot/genética , Manihot/virologia , Vírus de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Oxirredutases/genética
18.
PLoS One ; 14(8): e0220621, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31390343

RESUMO

Pathogen-free stocks of vegetatively propagated plants are crucial in certified plant production. They require regular monitoring of the plant germplasm for pathogens, especially of the stocks maintained in the field. Here we tested pre-basic mother plants of Fragaria, Rubus and Ribes spp., and conserved accessions of the plant genetic resources of Rubus spp. maintained at research stations in Finland, for the presence of viruses using small interfering RNA (siRNA) -based diagnostics (VirusDetect). The advance of the method is that unrelated viruses can be detected simultaneously without resumptions of the viruses present. While no virus was detected in pre-basic mother plants of Fragaria and Ribes species, rubus yellow net virus (RYNV) was detected in pre-basic mother plants of Rubus. Raspberry bushy dwarf virus (RBDV), black raspberry necrosis virus (BRNV), raspberry vein chlorosis virus (RVCV) and RYNV were detected in the Rubus genetic resource collection. The L polymerase encoding sequence characterized from seven RVCV isolates showed considerable genetic variation. The data provide the first molecular biological evidence for the presence of RYNV in Finland. RYNV was not revealed in virus indexing by indicator plants, which suggests that it may be endogenously present in some raspberry cultivars. In addition, a putative new RYNV-like badnavirus was detected in Rubus spp. Blackcurrant reversion virus (BRV) and gooseberry vein banding associated virus (GVBaV) were detected in symptomatic Ribes plants grown in the field. Results were consistent with those obtained using PCR or reverse transcription PCR and suggest that the current virus indexing methods of pre-basic mother plants work as expected. Furthermore, many new viruses were identified in the collections of plant genetic resources not previously tested for viruses. In the future, siRNA-based diagnostics could be a useful supplement for the currently used virus detection methods in certified plant production and thus rationalize and simplify the current testing system.


Assuntos
Vírus de Plantas/isolamento & purificação , RNA Interferente Pequeno , Rubus/virologia , Finlândia , Fragaria/virologia , Métodos , Vírus de Plantas/genética , Reação em Cadeia da Polimerase , Ribes/virologia
19.
Plant Physiol Biochem ; 142: 34-42, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31255907

RESUMO

The 24-kDa protein (p24) encoded by Grapevine leafroll-associated virus 2 (GLRaV-2) is an RNA-silencing suppressor (RSS), but its effect on active viral infection is unclear. Using a Potato virus X (PVX)-based expression system, we demonstrated that p24 elicits lethal systemic necrosis in Nicotiana benthamiana, sharing typical characteristics of the hypersensitive response (HR), and that NbRAR1 (a cytoplasmic Zn2+-binding protein) is involved in the PVX-p24-mediated systemic necrosis. Moreover, expression of p24 from Barley stripe mosaic virus (BSMV) vector triggered local necrosis in infiltrated patches of N. benthamiana, likely inhibiting viral systemic spread. By deletion analysis, we demonstrated that amino acids (aa) 1 to 180, which are located in the region (aa 1-188) previously shown to be necessary for p24's RSS activity, is sufficient for p24 to elicit systemic necrosis in the context of PVX infection. Using substitution mutants, we revealed that silencing-suppression-defective mutants R2A and W54A induce only a mild necrotic response; two mutants without self-interaction ability previously shown to lose or retain weak suppression function also displayed decreased pathogenicity: W149A without RSS activity elicited a mild necrotic response, whereas V162H/L169H/L170H which retains weak RSS activity was able to induce systemic necrosis, but with a 1- to 2-day delay. Taken together, p24 plays an important role in GLRaV-2 pathogenesis, triggering HR-like necrosis in N. benthamiana plants when expressed from PVX or BSMV vector; both the silencing suppression and self-interaction are crucial for p24's pathogenicity activity, and the region of p24 for determining systemic necrosis is mapped to aa 1-180.


Assuntos
Closterovirus/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Potexvirus/genética , Tabaco/virologia , Proteínas Virais/genética , Morte Celular , Closterovirus/patogenicidade , Regulação Viral da Expressão Gênica , Inativação Gênica , Interações Hospedeiro-Patógeno/genética , Mutação , Folhas de Planta/genética , Folhas de Planta/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Vírus de Plantas/patogenicidade , Potexvirus/patogenicidade , Tabaco/citologia , Proteínas Virais/metabolismo
20.
Curr Opin Insect Sci ; 33: 7-18, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31358199

RESUMO

Plant virus infection fundamentally alters chemical and behavioral phenotypes of hosts and vectors. These alterations often enhance virus transmission, leading researchers to surmise that such effects are manipulations caused by virus adaptations and not just by-products of pathology. But identification of the virus components behind manipulation is missing from most studies performed to date. Here, we evaluate causative empirical evidence that virus components are the drivers of manipulated host and vector phenotypes. To do so, we link findings and methodologies on virus pathology with observational and functional genomics studies on virus manipulation. Our synthesis provides an overview of progress, areas of synergy, and new approaches that will lead to an improved mechanistic understanding of host and vector manipulation by plant viruses.


Assuntos
Comportamento Animal , Insetos Vetores/virologia , Vírus de Plantas/fisiologia , Animais , Insetos Vetores/fisiologia , Insetos/fisiologia , Insetos/virologia , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de Plantas/patogenicidade
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